Swab Materials

Before choosing flocked nylon as the best material for Fisherly’s prototype, we referred to several research papers that discuss the characteristics of swab materials and their advantages.

According to research by Brujins et al., the differences in the morphology between flocked nylon, cotton and rayon swabs result in a significant difference in their retrieval and release performance^[1]. Flocked nylon swabs have short nylon fibre strands with no internal core attached perpendicularly to the plastic shaft. This trait allows for an increased sample uptake and release^[2], whereas rayon and cotton swabs, composed of longer fibres tightly wound on the shaft, have a lower retrieval performance^[1].

The efficiency of flocked nylon swabs is supported by a study conducted by Paula Väre, Klaus Hedman and Maija Lappalainen. Flocked nylon swabs showed a great releasing property of 70% to 80%, while cotton swabs released about 18% to 30% of the sample^[3]. In addition to high releasing efficiency, the short fibre strands make flocked nylon swabs more appealing. In contrast to rayon and cotton swabs, which leave some material and impurities due to their longer fibres, flocked nylon swabs barely leave any residues on the sample after swabbing^[1]. With these points in mind, we decided that flocked nylon swabs would be the most suitable to gather biogenic amines from fish samples.

Soaking Buffer

Dr. Peter Luk from the Agriculture, Fisheries, and Conservation Department mentioned that the moisture level of the fish surface differs among fish samples, depending on the type and storage condition. Hence, it is crucial to have a way to limit and standardise the sample collection area. After getting feedback from Dr. Luk, we decided to include a soaking buffer in our kit. This modification would create a more uniform moisture level on the swab during sample collection; having a standardised amount of soaking liquid for the swab will minimise the moisture level difference within samples.

Aspects that we considered when choosing the soaking buffer are food safety and its compatibility with the extraction buffer and the cell-free system. Of the five extraction buffers, MQ and citrate buffer were also candidates for soaking buffer, as they are food safe. However, after our CFS and extraction buffer compatibility experiment, we found that citrate buffer interferes with the performance of the CFS. Moreover, we could not gather enough data to deduce whether or not the mixing of two different buffer chemicals compromises the bioamines collected, hinder the extraction efficiency, or affect the biosensor. Therefore, we decided to use MQ as the soaking solution to minimise this concern.

The specific volume of the soaking volume was based on the maximum volume the swab can hold, which is 220 μL. The details of deriving 220 μL can be found in week 12 notebook.. Since we did not want the soaking buffer to be present in excess to leave a lot of moisture on the surface, we decided on using 50 μL, which is around 20%, in our experiments.

Extraction Buffer

The extraction efficiency of the extraction buffer is one of the important factors in our prototype, as it affects the recovery rate of bioamine samples from the swab during sample processing. Therefore, several candidates were considered. Five were chosen based on research:

1. Citrate Buffer
   Citrate buffer was chosen as one of the candidates after reading a paper that used citrate buffer at different pHs to extract bioamines via capillary electrophoresis with pulsed amperometric detection^[4].


2. Hydrogen chloride (HCl)
   Several papers that studied the bioamine concentration via the High-Performance Liquid Chromatography (HPLC) method mentioned HCl as a suitable option for bioamine extraction. According to research that worked with bioamines in Lycium barbarum L., HCl was superior to other extraction reagents, such as perchloric acid and trichloroacetic acid^[5]. Another study that used cheese samples used HCl as well, with the reasoning that it is a simple and inexpensive option.


3. Trichloroacetic acid (TCA)
   Trichloroacetic acid is a popular extraction solution, often used in High-Performance Liquid Chromatography to quantify bioamine levels. According to research, TCA is highly recommended to extract bioamines from fish or meat samples as they show a satisfactory recovery rate^[6].


4. Water
   Water, without any additional chemicals or treatments, was used to extract bioamines from fish muscle tissues in a paper that discussed using ion-exchange chromatography coupled with spectrometry detection^[7]. Although they had an additional separation step after bioamine extraction, the sample of interest and the method of sample extraction were the same. Therefore, water was chosen as one of the extraction buffers.


5. Sodium tetraborate
   A commercialised histamine test kit from the Kikkoman Biochemifa Company used sodium tetraborate as the reagent for sample extraction^[8]. Although we failed to get a response to having a meeting to discuss their experience with testing different extraction reagents and seek advice on Fisherly’s current prototype design, this extraction reagent was added as one of the options to test out.

Before directly comparing the extraction efficiency through the swab recovery rate experiment, the cell-free system (CFS) and extraction buffer compatibility experiment was performed first to eliminate ones that hinder the cell-free system’s function. Through a series of selection processes, the final extraction buffer was chosen. (A more detailed explanation about the selection of buffers can be found here.)

The concentration of the extraction buffer is set as 0.01M so that a minimum 1:1 molar ratio of extraction buffer to bioamines could be achieved at 1000 ppm, which is the upper limit of our biosensor that will give a red output.

Moreover, in order to adjust the activation time of the cell-free system so that it gives accurate colourimetric outputs at desired bioamine concentrations, a basal level of 192 ppm histamine is included in the extraction buffer, so that our biosensor will produce red output after 23 minutes. More details and calculations can be found here.

Filter

The fish surface is not entirely sterile and contains potential sample contaminants. Therefore, we decided to include a filter in the sample outlet zone to reduce the risk of introducing bacterial contamination to our cell-free system.

We considered the following four criteria when choosing the filter material candidates: mechanical strength, hydrophilicity, protein binding capacity, and leachability.

• Two major types of filters are available in the market, hydrophobic and hydrophilic. While hydrophobic membrane filters are ideal for air and gas filtration, they are not suitable for filtering aqueous solutions. On the other hand, hydrophilic membrane filters are commonly used for water-based fluids. Since the extraction buffer in our kit is water-based, the hydrophilic ones are more suitable.
• The most common pore size used to filter out bacteria is 0.22 μm. Filters with small pores are prone to pore clogging if the material has high reactivity with protein. As bioamines are smaller than protein, we wanted to ensure that bioamines can pass easily through the pores. Therefore, a filter with low protein binding capacity is needed.
• Leachability refers to the ability of the compounds in the filters to leach from the filter materials. As our goal of using a microfilter is to get rid of contaminants, it would defeat its purpose if the filter itself contributes to contamination. We compared the three following candidates based on the five mentioned criteria:

1. Nylon
   Nylon is naturally hydrophilic and has high mechanical strength^[9]. However, after further research, we found that nylon demonstrates extremely high protein adsorption compared to the other candidates^[10].


2. Hydrophilic polyvinylidene difluoride (H-PVDF)
   PVDF is commonly used for liquid microbiological analysis. It has a wide range of chemical compatibility, high bacterial filtration efficiency, good mechanical strength, low leachability, and low protein adsorption^[10][11][12]. Unfortunately, PVDF is naturally hydrophobic, which means it requires a hydrophilicity treatment to be used in our product.


3. Polyethersulfone (PES)
   PES is compatible with most chemicals, robust, and hydrophilic. PES shares similar qualities with PVDF in terms of bacterial filtration efficiency, protein adsorption and leachability but has a lower price^[11]. Therefore, it is a highly suitable option.

After a thorough comparison, we concluded that the PES filter is the most appropriate for our product.

Rehydration volume

In order to identify the best rehydration method for the freeze-dried cell-free biosensor, we consulted Professor Taishi Tonooka from the faculty of Mechanical Engineering, Kyoto Institute of Technology, who has experience in incorporating cell-free systems into different devices. He suggested using the volume lost during the freeze-drying process to rehydrate the cell-free system. In our case, the volume of our cell-free biosensor is 50 μl, which means that 50 μl of the processed sample would be suitable as the rehydration volume.

Storage condition and time

In order to determine the storage condition and duration of our product, we looked into various research papers to compare the efficacy of non-lyophilized CFS and lyophilized CFS after rehydration at different temperatures and duration.

According to research that looked into cell-free biosensors for biomedical applications, while non-lyophilised CFS showed rapid degradation in efficacy, freeze-dried ones could be preserved at various temperatures, −80°C, 20°C, 4°C, or 25°C for up to one year, although an 80% decrease in signal was observed after the rehydration of these components^[16].

Moreover, another study by Salehi et al. looked into the lyophilized CFS and non-lyophilized CFS sensor stability when stored at various temperatures, from −80°C to 25°C for up to 1 year. It revealed that non-lyophilized extracts have a rapid decrease in efficacy, especially at higher temperatures, whereas the lyophilized extracts were more stable^[17]. However, the freeze-dried CFS was also affected by temperature, with its viability decreasing more rapidly at higher temperatures. The lyophilized extracts also lost activity after 1 year.

Therefore, to ensure product quality, the suitable storage temperature and viability time were decided to be 4 degrees and 90 days.